DESCRIPTION (Verbatim from the applicant's abstract) The overall goal of this project is to understand the in vivo function of histones and their role in nuclear differentiation. These studies are greatly facilitated by the remarkable nuclear dimorphism of Tetrahymena in which transcription occurs specifically in vegetative somatic macronuclei and mitosis and meiosis occurs only in germline micronuclei. The investigators are performing in vivo analyses of histone function our newly developed methods for mass transformation and gene replacement. There are 10 distinct histone types in T thermophila. These are encoded in 12 genes, all of which have now been cloned and sequenced. Tetrahymena thermophila is unique in having the only completely cloned and sequenced histone gene complement containing linker histones, a conserved minor H2A.F/Z variant and a basal H3.3 variant as in mammalian cells. T. thermophila may also be unique in the depth to which the histone primary sequence variants and secondary modification sites have been characterized and the degree to which these have been associated with nuclear processes in different physiological and developmental states. The small gene number coupled with gene replacement in both somatic and germline nuclei should now enable novel, in vivo tests of hypotheses emerging from previous studies. Concentrating on studies of the phosphorylation of linker histones, which they demonstrated to regulate gene expression in vivo, both positively and negatively will address the mechanism of these effects. Similarly, he will address the mechanisms by which H3 phosphorylation acts to affect mitotic and meiotic chromosme segregation and condensation. He will analyze the function of phosphorylation of and ubiquitination of H2A, conserved histone modifications, whose function is unknown. They are testing the hypothesis, in both yeast and Tetrahymena, that the conserved H2A, F/Z variant functions to activate the expression of a distinct subset of genes. Finally, they are testing the hypothesis that site-specific acetylation and other modifications serve as a "histone code' to regulate chromatin function. Given the conservation of histones and their secondary modifications, and their demonstrated importance in disease, they expect the studies proposed here to provide important insights into the functions of linker histones, histone variants and histone modification in transcription and chromosome condensation in eukaryotes.